Global consumption of wood products is projected to increase 25% over current levels by 2015 (McLaren 1999). Full citations for the references cited herein are provided before the claims. Forest plantations are increasingly important to meet these global demands because their faster growth rates result in much more harvestable volume per unit area than natural forests (Hagler 1996, Sedjo 1999). Thus, reliance on plantations reduces the need to harvest natural forests, allowing them to be used for other societal purposes. In fact, as little as 5 to 10% of the total area of world's forests would be required to meet global demands for wood products if this area were devoted to fast-growing plantations (Hagler 1996, Sedjo and Botkin 1997). Further, the faster growth rates mean high rates of carbon sequestration that may mitigate the effects of global warming. These facts, coupled with the declining area available for commercial forest harvests due to deforestation and government restrictions, have led to a global effort to increase plantation growth rates per unit area above current values through both classical and new technologies (Fox 2000).
Viewed as an agricultural crop, timber is the single highest-valued crop in the USA and loblolly pine (Pinus taeda L) is the most important commercial tree species in the USA. Each year more than 900 million seedlings are used to establish loblolly pine plantations on more than half a million hectares (Pye et al. 1997). The total acreage of the loblolly pine plantation estate is estimated at more than 12 million hectacres (Byram et al. 1999). Loblolly pine is also important for its ecological and biological importance in native forests. Its native range spans 14 states from southern New Jersey south to central Florida and west to Texas. In these natural forests it is the dominant tree species on 11.7 million ha (Baker and Langdon 1990). Thus, loblolly pine is nearly equal in its distribution between native and planted forests totaling 23.7 million hectares. By comparison, the total expanse of plantations of hybrid poplar in the Pacific Northwest is approximately 25,000 ha (Nuss 1999), which is only 0.2% of the area planted in loblolly pine.
Due to its overwhelming commercial importance, tree breeding programs for loblolly pine began in the 1950's, and virtually all forest products companies and state agencies are involved in genetic improvement programs (more than 30 organizations) (Byram et al. 1999, Li et al. 1999). These programs have used classical methods of selection, genetic testing and breeding to make demonstrable genetic progress. Unfortunately the progress is hindered, compared to that in agricultural crops, by the large size and long-lived nature of pines (eight years in field tests to make selections followed by another five or more years to complete breeding). For these reasons, most loblolly pine programs are only in their second or third cycle of breeding after nearly 50 years, when in some crops more than one cycle is completed in a single year.
Loblolly pine (Pinus taeda L.) is the most intensively grown tree species in the USA for pulp and solid wood products with plantations exceeding 12 million hectares. The extraction of lignin from wood during the production of pulp and paper requires the use of costly chemicals that are toxic to the environment. Significant progress towards increasing pulping efficiency has been achieved in poplar through the genetic manipulation of genes involved in lignin biosynthesis (Baucher et al., 1996, Hu et al., 1999; Pilate et al., 2002). One of the key enzymes successfully targeted, cinnamyl alcohol dehydrogenase (CAD), catalyzes the final step in the synthesis of monolignols by converting cinnamaldehydes to cinnamyl alcohols. Field-grown transgenic poplar with reduced-CAD allowed easier delignification, using smaller amounts of chemicals and yielded more high quality pulp without an adverse effect on growth (Pilate et al., 2002).
A null CAD allele (cad-n1) has been discovered in the loblolly pine clone 7-56 which is heterozygous for the null allele (MacKay et al., 1997). Homozygous seedlings (cad-n1/cad-n1) obtained by selfing, contain between 0-1% of wild type CAD activity (MacKay et al., 1997) and display a brown-red wood phenotype. The expression level of cad transcript in shoot, megagametophyte and xylem tissues was 20 times less in cad-n1 homozygous plants compared to wild type (MacKay et al., 1997).
Deficiency of CAD in cad-n1 homozygotes only slightly reduces lignin content but drastically alters lignin composition (MacKay et al., 1997; Ralph et al., 1997; Lapierre et al., 2000; MacKay et al., 2001). The major lignin composition change was attributed to the incorporation of dihydroconiferyl alcohol (DHCA), a minor component of most lignins, but elevated to levels 10-fold higher in cad-n1 homozygous trees. Coniferaldehyde, the substrate of CAD, and vanillin are also present in increased levels while the coniferyl alcohol component of normal lignin decreased.
The mutation has a variable effect on pulping efficiency, depending on the age of the trees and whether the mutation is present in a homozygous or heterozygous state. In totally CAD-deficient trees (cad-n1/cad-n1), delignification was significantly easier but the pulp yields were relatively low (˜33%) compared to normal trees (48%) (Dimmel et al., 2001). In 4-6 year old partially CAD-deficient trees (heterozygous) delignification increased in efficiency by ˜20% and yields were similar to wild type (Dimmel et al., 2002). In contrast to these younger trees, a small sample of 14 year old partially CAD-deficient trees displayed no major differences in ease of delignification and pulp yield (Dimmel et al., 2002).
In addition to lignin composition changes, the cad-n1 allele appears to be associated with increased stem-growth traits in heterozygous trees (Wu et al., 1999). This growth promotion correlates to an increase in debarked volume of 4-year old trees (14%) (Wu et al., 1999) that is also observed in 14-year old trees (Dimmel et al., 2002). A likely explanation could be that trees harboring the cad-n1 allele may invest fewer resources into the production of monolignols, allowing reallocation of resources towards growth. Promotion of growth was also observed in transgenic poplar with the lignin biosynthetic enzyme 4-coumarate:coenzyme A ligase (4CL) down-regulated (Hu, et al., 1999).
For the above reasons, it is desirable to be able to select pine trees that harbor the null CAD allele (cad-n1). Traditionally, the mutation has been diagnosed using CAD isozyme analysis on haploid megagametophytes obtained from seed or by using genetic markers closely linked to the mutation (MacKay et al., 1997). These prior art methods are slow and tedious. It takes numerous years for pine tree seedlings to produce suitable seed for CAD isozyme marker analysis. In addition, linked genetic marker analysis is slow and often yields inaccurate results. There is thus a tremendous need to develop methods that allow rapid and accurate identification of pine trees that harbor the null CAD allele (cad-n1).